ANTIMUTAGENIC AND ANTIOXIDANT ACTIVITY OF THE EXTRACT FROM BELAMCANDA CHINENSIS (L.) DC.

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1 Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 63 No. 3 pp. 213ñ218, 2006 ISSN Polish Pharmaceutical Society ANTIMUTAGENIC AND ANTIOXIDANT ACTIVITY OF THE EXTRACT FROM BELAMCANDA CHINENSIS (L.) DC. DOROTA WOèNIAK A, JAN OSZMIA SKI B and ADAM MATKOWSKI A * A Department of Pharmaceutical Biology and Botany, Medical University in Wroc aw, Al. Jana Kochanowskiego 10, Wroc aw, Poland, B Department of Fruit, Vegetable & Herb Technology, Agricultural University of Wroc aw, C.K. Norwida 25/27, Wroc aw, Poland Abstract:The antioxidant and antimutagenic properties of the extract from the oriental natural drug ñ Belamcanda chinensis (L.)DC. (Iridaceae) rhizomes have been examined. Three different antioxidant assays and Salmonella antimutagenic test have been used. The extract has strong free radical scavenging activity as shown by the DPPH assay with EC 50 of 63.4 µg/ml. Similarly, the high ability to reduce the transition metal ions was shown by phosphomolybdenum assay as well as effective prevention of hydroxyl radical induced linoleic acid peroxidation. The latter averaged 77.9% with an EC 50 of 45 µg/ml. The mutations induced in two Salmonella typhimurium strains (TA98 and TA100) by both directly (NQNO) and indirectly acting (2AF) chemical mutagens were inhibited efficiently in dose dependent manner for concentrations of µg/ml. The inhibition percentage in TA98 was 62.8% for direct mutagen and 94.4% for enzymatically activated promutagen. In TA100 the inhibition was 82.7% and 73.5%, respectively. Keywords: antioxidant, Belamcanda, mutagenesis, lipid peroxidation, isoflavonoid The numerous environmental and endogenous mutagens and carcinogens may act through the generation of reactive oxygen species (ROS). The generation of ROS is also associated with environmental pollution, UV radiation and several normal metabolic processes. ROS may also play a major role as endogenous initiators of degenerative processes, such as DNA damage (1). Dietary intake of antioxidants could be an important factor in the bodyís defense mechanisms against ROS and carcinogens because many antioxidants are being identified as anticarcinogens (2). Flavonoids (flavones, flavonols, flavanones and isoflavones) have very effective pharmacological properties with respect to antioxidation and anticarcinogenesis (3) but despite having a related structure, not all of the flavonoids do show the same mechanism of action. Some flavonoids have been reported to act as scavengers of various oxidizing species or as metal chelators. Isoflavonoids showed the relatively strong activity against lipid peroxidation (4). In our studies, we search for natural antioxidants and radical scavengers, which may have a direct or indirect correlation with chemoprevention of human degenerative disorders. The extensive resources of the traditional Oriental phytotherapeutic systems should be utilized and there is a need for comprehensive research on the species which are likely to gain more popularity in other parts of the world. Belamcanda chinensis (L.) DC. (Iridaceae) is a perennial growing on the warm hill sides in South- East Asia including Japan and the Korean peninsula. Its underground parts have been used as Chinese traditional medicine to treat a variety of inflammation related diseases, as well as an inhibitor of some common skin fungi (5,6). One of the potential mechanisms of its action may involve antioxidant and genoprotective properties that are important for the prevention of many degenerative diseases. Little is known about the properties of flavonoid rich extracts from rhizomes of Belamcanda chinensis with respect to the aforementioned activities. Only few isolated compounds have been studied, including the cancer-preventive and phytoestrogenic activity (7-11) and the potential of the complex extract as well as the mechanisms involved in the presumed genoprotective activity need to be examined. As for the chemical constituents of the plant, the occurrence of iridal-type triterpenoids and isoflavonoids, as well as xanthones, stilbenes and aurones in the rhizomes has been reported in the literature (12,13). The main compounds from isoflavonoids are tectoridin, iridin and their aglycones tectorigenin, irigenin and others like irisflorentin, rhamnocitrin, belamcandin as well as their derivatives differing in substitution patterns (6,14-17). The present work describes for the first time the antioxidant and antimutagenic activity of the methanolic extract of dried rhizomes of cultivated Belamcanda chinensis. The antimutagenic activity has been assayed in the 213

2 214 DOROTA WOèNIAK et al. Ames test and the antioxidant and free radical scavenging activities have been studied in three spectrophotometric assays which test different aspects of antioxidation. EXPERIMENTAL Plant material The plants of Belamcanda chinensis were obtained from the collection of the Medicinal Plants Botanical Garden in Wroc aw where this species has been successfully acclimated and grown for several seasons. The underground parts were harvested in October, washed and air-dried at 40 C. The dried rhizomes and roots (60 g) were chopped and macerated in 600 ml Me-OH for 5 days followed by reflux-extraction at 60 C. The crude extract has been evaporated under vacuum at 40 C and redissolved in ethanol for bioactivity analysis. The extract yield was 19.4% w/w. Reagents DPPH, Trizma base, NADP, 2AF (2-aminofluorene), NQNO (4-nitroquinoline-N-oxide), DMSO, G-6-P (glucose-6-phosphate), thiobarbituric acid (TBA) and ψ-tectorigenin were purchased from Sigma-Aldrich (PoznaÒ, Poland), Linoleic acid was from Fluka AG, trichloroacetic acid from Ubichem UK, Bacto-Agar from Beckton Dickinson France (Le Pont de Claix, France), Oxoid Nutrient Broth No.2 from Oxoid (Basingstoke, UK). All other reagents and solvents were obtained from POCh, (Gliwice, Poland). The S9 fraction was purchased from In Vitro Technologies (Catonsville, MD, U.S.A). Antioxidant tests DPPH radical anion scavenging activity Scavenging of free radicals in vitro has been evaluated with DPPH free radical assay according to Brand-Williams et al. (18). Briefly, 200 µl of the dissolved extract in different concentrations ( µg/ml) was added to 1.8 ml of the MeOH-DPPH (100 µm) solution. The absorbance at 517 nm was measured 5 s and 30 min after mixing using the Shimadzu 1601 UV/VIS spectrophotometer (Shimadzu Corp., Kyoto, Japan) against a blank containing an appropriate extract dilution without DPPH. The percentage of scavenged DPPH was then calculated for both incubation periods. In addition, for 30 min incubation, the antiradical activity was expressed as the EC 50 value defined as the concentration of extract at which 50% of maximum scavenging activity was recorded. Phosphomolybdenum reduction assay The antioxidant capacity of the extract was assessed as described by Prieto et al. (19). The extract in dilutions from 5 µg/ml to 500 µg/ml was combined with the reagent solution containing ammonium molybdate (4 mm), sodium phosphate (28 mm) and sulfuric acid (600 mm). The reaction mixture was incubated in a water bath at 37 C or 90 C for 90 min. The absorbance of the colored complex was measured at 695 nm. The antioxidant activity was compared to that of ascorbic acid and quercetin in the same concentration range. Inhibition of the linoleic acid peroxidation The procedure of Choi et al. (20) using a Fenton reaction induced lipid peroxidation has been adapted for this assay. The Belamcanda chinensis extract in concentrations range of µg/ml has been mixed with 300 µl 50 mm TRIS-HCl buffer, ph 7.5, 500 µl of 20 mm linoleic acid and 100 µl of 5 mm ascorbic acid. The peroxidation was started with addition of 100 µl 4 mm FeSO 4. The reaction mixture was incubated for 60 min. at 37 C. Thereafter, 2 ml of 10% ice cold trichloroacetic acid was added and 1 ml aliquots of the samples was added with 1 ml of 1% thiobarbituric acid. The TBA/sample mixture was heated in the water bath at 95 C for another 60 minutes. The absorbance was read at 532 nm and the percentage of linoleic acid peroxidation inhibition was calculated as in Choi et al. (20) using appropriate controls. Quercetin was used as a positive control in all antioxidant assays. Antimutation assay The mutation test was carried out according to the Ames method (21). The test mixtures contained: 0.1 ml of the extract (in doses 50 ñ 500 µg per plate), 0.1 ml overnight culture of Salmonella typhimurium TA98 or TA100 strains, 0.1 ml of standard mutagen (2-aminofluorene, 5 µg/plate or 4-nitroquinoline-Noxide, 0.5 µg/plate, in DMSO). In the case of 2AF, 0.5 ml of S9 fraction with NADP and G(6)P as cofactors, and in the case of NQNO 0.5 ml phosphate buffer of ph 7.4 was added. The test tubes with promutagen were preincubated at 37 C for 20 min. After mixing with top agar the samples were poured onto minimal glucose agar plates, in three repetitions. The cytotoxicity of tested compounds was assayed with S. typhimurium TA98 and TA100 strains in accordance with Maron and Ames (22). Chromatography The extract was preliminary screened for flavonoid compounds using TLC and HPLC. For

3 Antimutagenic and antioxidant activity of the extract from Belamcanda chinesis (L.) DC 215 TLC the Merck SG 60 10x20 cm glass plates were used eluted in horizontal DS-9 chambers (Chromdes, Lublin, Poland) with 3 different mobile phases. The chromatograms have been examined for putative isoflavonoid spots (23) under UV (254 nm and 366 nm) illumination before and after derivatization with ammonia fumes or aluminum chloride spraying. Reversed phase DAD-HPLC was performed with Merck-Hitachi L-7455 device with L HPLC pump and D-7000 HSM multisolvent delivery system using the PLRP-S 100 Å 250x4,6 mm (5 µm) column (Polymer Laboratories). Gradient elution with 80% acetonitrile in 4,5% formic acid (B) and 4.5% formic acid (A) at the flow rate of 1 ml/min. has been programmed as follows: 0-16 min. (A), min 20% A and 80% B, min. 100% B. The peaks were registered at 260 nm and the DAD spectra for each peak have been recorded between nm. 5,7,4í-trihydroxy-8- methoxyisoflavone (y-tectorigenin) was used as a calibrator standard for quantitation. Statistical analysis Each of the antioxidant and antimutagenic assays was performed in triplicate. The results obtained in the Ames test (mean ± SD, n = 8) were analyzed for statistical significance by Studentís t-test. The percentage of antimutagenic activity was calculated after subtraction of spontaneous revertant numbers. EC 50 values were calculated using nonlinear regression module of Statistica 5.0. (Statsoft, Poland). RESULTS The direct antioxidant action has been assayed by scavenging of free radicals. This kind of antioxidative properties was tested by the DPPH radical decolorization assay. The Me-OH extract of Belamcanda chinensis exhibited dose dependent free radical scavenging activity in the DPPH assay with the EC 50 of 63.4 µg/ml (Fig.1). Moreover, in higher concentrations ( µg/ml) the free radical scavenging was noticeable as early as in the 5 th second of incubation. We have also used the extract of Belamcanda chinensis to determine the antioxidant capacity by the formation of phosphomolybdenum complexes. This method is based on the reduction of Mo(VI) to Mo(V) by the antioxidant compounds and the formation of a green Mo(V) complex with a maximal absorption at 695 nm. This assay also revealed a relatively potent reducing power of the Belamcanda chinensis roots (Table 1). In the concentration range of µg/ml the Me-OH extract showed strong and dose dependent antioxidant capacity. Some reducing activity of Belamcanda chinensis was observed from the lowest (5 µg/ml) concentration tested while it became visibly stronger above 12 µg/ml increasing exponentially till the highest (500 µg/ml for 40 C and 250 µg/ml for 90 C) concentration. Results of the linoleic acid peroxidation assay are shown on Fig.2. The maximum mean inhibition reached 77.9 % (±14.1) for the highest extract concentration (500 µg/ml), while the maximum observed inhibition among six sample replicates was as high as 94.9%. The calculated IC 50 of Belamcanda chinensis extract for inhibition of TBARS production (product of lipid peroxidation) from Fenton system with Fe 2+ was 45 µg/ml. The crude extract of the Belamcanda chinensis rhizomes markedly and dose-dependently decreased the mutagenic activity of direct (NQNO) and indirect (2AF with S9 promutagen activating fraction) mutagens in the Ames test with the TA98 and TA100 Salmonella typhimurium strains (Table 2). Highest inhibition of NQNO activity by 62.8% and of 2AF by 94.4% with the TA98 strain and inhibition of NQNO activity by 82.7% and of 2AF by 73.5% in the case of TA100 strain was notified. This is significant antimutagenicity in the NQNO induced test as well as in the 2AF induced one. This may indicate that the extract can inhibit microsomal S9 enzymes activity as well as directly protect DNA molecule from the chemical mutagen. The extract did not exhibit mutagenic activity in the dose range of µg/ml, either in the presence or absence of the S9 promutagen activating fraction. We also noticed the non-toxicity of the extract to bacterial cells at the same doses tested in this study. DISCUSSION To answer how the extract as a putative antioxidant is supposed to act we were assaying it in three different antioxidant tests. Our results suggest that the direct free radical scavenging of the extract measured with DPPH, should suffice to exert a protective effect against ROS. The DPPH scavenging activity has been also demonstrated for some isoflavonoids isolated from the rhizomes of a related specie ñ Iris germanica L. (24). The results obtained in the phosphomolybdenum assay confirm high reducing potency of the Belamcanda chinensis extract towards the transition metal ions. The reducing power was compara-

4 216 DOROTA WOèNIAK et al. ble (Table 1) to the recognized hydrophilic antioxidant ascorbic acid, and to the pure flavonol quercetin. On the other hand, results from Fenton system with Fe 2+ imply that this extract might be exerting its protective influence on polyunsaturated fatty acids like linoleic acid through chelation of metal ions or by altering iron redox chemistry. Yokazawa et al. (25) described scavenging and antioxidation ability of 24 pure flavone compounds with the selectively generated radicals induced by Fe 2+ Fenton type reaction. Some flavones with stronger protecting effect against lipid peroxidation had the IC 50 between 2 ñ 46.3 µg/ml but most of them showed considerably weak action with IC 50 values about 300 µg/ml and even up to 500 µg/ml. In comparison to that data, the IC 50 estimated from sigmoid regression curve of Belamcanda chinensis extract for inhibition of TBARS production in Fenton system with Fe 2+, was 45 µg/ml, which places it as a relatively potent antioxidant. Lipid peroxidation is started by adding Figure 1. The DPPH scavenging activity of different extract concentrations measured at 515 nm. The results from measurements at 5 seconds (white bars) and 30 minutes (grey bars) after mixing the sample with DPPH solution. The whiskers indicate standard deviation of 4 measurements. Table 1. Results of the phosphomolybdenum antioxidant assay of B. chinensis Me-OH extract. The reducing power of different extract concentrations is expressed as ascorbic acid or quercetin mass and molar equivalents. Ascorbic acid equivalents (± SD) Quercetin equivalents (± SD) extract 40 C 90 C 40 C 90 C [µg/ml] µg µm µg µm µg µm µg µm ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±292.5 * * ± ±3.3 * * *The results from 90 C incubation at 500 µg/ml are omitted because the absorbance was out of range (over 3.99 units) Table 2. Inhibitory influence of Belamcanda chinensis MeOH-extract on the mutagenicity of NQNO and 2AF mutagens in the Ames test with Salmomella typhimurium strains TA98 and TA100. Numbers of revertant colonies ± SD. Salmonella typhimurium TA98 strain TA100 strain fraction S9 -S9 +S9 -S9 +S9 control* 20 ± 3 32 ± ± ± 12 mutagen** 428 ± ± ± ± 35 Me-OH extract [µg/ml] ± ± ± ± ± ± ± ± ± ± ± ± ± ± 11 *control: spontaneous revertant number per plate **revertant numbers induced by mutagens: NQNO direct acting (-S9) or 2AF indirect acting (+S9)

5 Antimutagenic and antioxidant activity of the extract from Belamcanda chinesis (L.) DC 217 Figure 2. Inhibition percentage of linoleic acid peroxidation by the Belamcanda chinensis extract. The bars show mean inhibition (n=6, with SD whiskers). metal ions, e.g. Fe 2+ and ascorbate. In these cases, the antioxidant effect could occur not only by peroxyl radical scavenging but also by metal ion chelation. In order to examine the quality of the methanolic extract and to obtain an insight into the potentially active phenolic phytochemicals, a preliminary chromatographic analysis has been performed using TLC and HPLC. The presence of several putative isoflavonoid compounds (23) has been confirmed with accordance to the literature data (6,14,16) (Fig. 3). The total quantity of isoflavonoids in the extract has been estimated by HPLC as mg/g of the dry extract. However, it has to be pointed out that the use of one compound only as internal standard might have resulted in over- or underestimation due to the differences in response factors between the reference compound and the individual constituents of the extract. Oxidative damage to the DNA, proteins and membrane lipids has been postulated to be a major but not the only mechanism leading to aging and degenerative diseases (1,2). Reactive oxygen Figure 3. An HPLC fingerprint of Belamcanda chinensis MeOH extract recorded at 260 nm. Arrows indicate the isoflavonoid peaks identified from their DAD-UV spectra.

6 218 DOROTA WOèNIAK et al. species (ROS) and lipid peroxidation products can also give rise to mutagenic changes in the DNA. In addition to the protective effects of endogenous enzymatic defense mechanisms, consumption of exogenous natural antioxidants appears to be of great importance. In the present study, we observe both the antioxidant capacity and antimutagenic activity of the extract from rhizomes of Belamcanda chinensis but it is the total activity of various bioactive compounds present in the extract. The hypothetical correlation between antioxidant and antimutagenic properties needs further mechanistic studies to be ultimately confirmed. In the case of natural products, it is commonly found that a mixture of compounds as the Me-OH extract have much more activity than a separated compound. The complex mix of components may be capable of function in a variety of ways and there are both synergistic and antagonistic interactions possible. Future work on the bioactivity guided fractionation is required for the isolation and characterization of individual flavonoids from this extract and to establish the mechanisms involved in the antioxidant capacity and antimutagenic effects of compounds from Belamcanda chinensis. A potential role for other, non-phenolic constituents of belamcanda also has to be determined. The thorough knowledge of the antioxidant and genoprotective properties will promote wider recognition of this valuable oriental plant in phytotherapy. Acknowledgements We thank Katarzyna Szepielak and Fatima Sadek for their valuable assistance, as well as Mrs. Agata Biernat, M.Sc. from the Medicinal Plants Garden for making enough plant material available for the study. The research was partially supported by the University Research Grant No. 1120/2004 REFERENCES 1. Halliwell B., Gutteridge J.M.C.: Free Radicals in Biology and Medicine. Oxford University Press, Oxford Ames B.N., Shigenga M.K., Hagen T.M.: Proc. Natl. Acad. Sci. USA 90, 7915 (1993). 3. Suschetet M., Siess M.H., Le Bon A.M., Canivenc-Lavie M.C.: Polyphenols 96 ñ Les Colloques 87 (1996). 4. Harborne J.B., Williams C.A.: Phytochemistry 55, 481 (2000). 5. ZhuY.P.: Chinese Materia Medica. Harwood Academic Publisher, Amsterdam 1998, pp Shin K.H., Kim Y.P., Lim S.S., Lee S., Ryu N., Yamada M., Ohuchi K.: Planta Med. 65, 776 (1999). 7. Wang Q.L., Lin M., Li, G.T.: Jpn. J. Pharmacol. 87, 61 (2001). 8. Jung S.H., Lee Y.S., Lee S., Lim S.S., Kim Y.S., Ohuchi K., Shin K.H.: Planta Med. 69, 617 (2003). 9. Seidlov -Wuttke D., Hesse O., Jarry H., Rimoldi G., Thelen P., Christoffel V., Wuttke W.: Phytomedicine 11, 392 (2004). 10. Jung S.H., Lee Y.S., Lim S.S., Lee S., Shin K.H., Kim Y.S.: Arch. Pharm. Res. 27, 184 (2004). 11. Thelen P., Scharf J.G., Burfeind P., et al.: Carcinogenesis 26, 1360 (2005). 12. Seki K., Haga K., Kaneko R.: Phytochemistry 38, 703 (1995). 13. Takahashi K., Hoshino Y., Suzuki S., Hano Y., Nomura T.: Phytochemistry 53, 925 (2000). 14. Yamaki M., Kato T., Kashihara M., Takagi S.: Planta Med. 56, 335 (1990). 15. Woo W.S., Woo E.H.: Phytochemistry 33, 939 (1993). 16. Ito H., Onoue S., Yoshida T.: Chem. Pharm. Bull. 49, 1229 (2001). 17. Jung S.H., Lee Y.S., Lim S.S., Lee S., Shin K.H., Kim Y.S.: Arch. Pharm. Res. 25, 306 (2002). 18. Brand-Williams W., Cuvelier M.E., Berset C.: Lebensmitt. Wiss. Technol. 28, 25 (1995). 19. Prieto P., Pineda M., Aquilar M.: Anal. Biochem. 269, 337 (1999). 20. Choi C.W., Kim S.C., Hwang S.S., et al.: Plant. Sci. 163, (2002). 21. Ames B.N., McCann J., Yamasaki E.: Mutat. Res. 31, 347 (1975). 22. Maron D., Ames B.N.: Mutat. Res. 113, 173 (1983). 23. Mabry T.J., Markham K.R., Thomas M.B.: The Systematic Identification of Flavonoids. Springer Publishing, New York Wollenweber E., Stevens J.F., Klimo K., Knauft J., Frank N., Gerhauser C.: Planta Med. 69, 15 (2003). 20. Yokazawa T., Dong E., Liu Z.W., Shimizu M.: Phytother. Res. 11, 446 (1997). Received: